Software systems and methods for predicting the melting temperature (Tm) and other characteristics of oligonucleotides, including modified oligonucleotides. Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.
Legal claims defining the scope of protection, as filed with the USPTO.
1. A method of predicting the melting temperature (T m ) of an oligonucleotide sequence, comprising: inputting into a computer system data representing a sequence of N bases of an identified oligonucleotide having N-1 neighboring base pairs; calculating in the computer system a (T m ) of the identified oligonucleotide sequence using the nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence, wherein said identified sequence includes at least one modified base selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases; and providing an output to a display or a user using the processing module, wherein said output represents a T m of the identified oligonucleotide sequence.
2. The method of claim 1 , wherein inputting includes receiving a polynucleotide sequence from GenBank.
3. The method of claim 1 , wherein inputting includes receiving a polynucleotide sequence and identifying one of a portion of the received sequence and a sequence complementary to a portion of the received sequence.
4. The method of claim 1 , wherein said at least one modified base includes a member selected from the group consisting of a base attached to an amino acid and a locked nucleic acid sugar.
5. The method of claim 1 , wherein said at least one modified base includes a member selected from the group consisting of PPA (4-amino-1H-pyrazolo [3,4-d]pyrimidine), PPG (6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one), PPPA (4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine), PPPG (6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one), PU (5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione), PC (6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one), HOPU (5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuU (5-(4-hydroxybut-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuC (6-amino-5-(4-hydroxybut-1-ynyl)-3-hydropyrimidine-2-one), HOBuPPA (4-(4-aminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH 2 ) 2 PPPA (3-prop-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino), (NH 2 ) 2 PPPAOH (2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol), (NH 2 ) 2 BuPPAOH (4-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH 2 ) 2 PPAI (4,6-diamino-3-iodopyrazolo[3,4-d]pyrimidine), (NH 2 ) 2 PPABr (3-bromo-4,6-diaminopyrazolo[3,4-d]pyrimidine), (NH 2 ) 2 PPACl (3-chloro-4,6-diaminopyrazolo[3,4-d]pyrimidine), PPAI (4-amino-3-(iodo)pyrazolo[3,4-d]pyrimidine), PPABr (3-bromo-4-aminopyrazolo[3,4-d]pyrimidine), PPAC1 (3-chloro-4-aminopyrazolo[3,4-d]pyrimidine) and HOBuPPG (6-amino-3-(4-hydroxybut-1-ynyl)pyrazolo[3,4-d]pyrimidin-4-ol).
6. The method of claim 1 , wherein said identified oligonucleotide sequence has a minor groove binder (MGB) attached thereto.
7. The method of claim 6 , wherein said MGB has a formula selected from the group consisting of: wherein the subscript m is an integer of from 2 to 5; the subscript r is an integer of from 2 to 10; and each R a and R b is independently a linking group to said modified oligonucleotide, H, —OR c , —NR c R d , —COOR c and —CONR c R d wherein each R c and R d is selected from the group consisting of H, (C 1 -C 12 )heteroalkyl, (C 2 -C 12 )heteroalkenyl, (C 2 -C 12 )heteroalkynyl, (C 1 -C 12 )alkyl, (C 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl, aryl(C 1 -C 12 )alkyl and aryl.
8. The method of claim 1 , further comprising: retrieving said nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence from a memory.
9. A computer readable storage medium including computer code for controlling a processor to calculate the melting temperature (T m ) of an oligonucleotide, the computer code including instructions that cause the processor to perform the following steps: input into a processing module data representing a sequence of N bases of an identified oligonucleotide having N-1 neighboring base pairs; retrieve nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence from a memory, wherein said identified sequence includes at least one modified base selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases; calculate a melting temperature (T m ) of the identified oligonucleotide sequence using the retrieved nearest neighbor thermodynamic parameters; and provide an output to a display or a user using the processing module, wherein said output represents a T m of the identified oligonucleotide sequence.
10. The computer-readable storage medium of claim 9 , wherein said input includes a Genbank code, wherein said instructions to identify include instructions to automatically retrieve a polynucleotide sequence from Genbank over a network using said input Genbank code.
11. The computer-readable storage medium of claim 9 , wherein said input includes a polynucleotide sequence.
12. The computer-readable storage medium of claim 9 , wherein said instructions to identify includes instructions to identify one of a portion of a received polynucleotide sequence and a sequence complementary to a portion of the received sequence.
13. The computer-readable storage medium of claim 9 , wherein said at least one modified base includes a member selected from the group consisting of a base attached to an amino acid and a locked nucleic acid sugar.
14. The computer-readable storage medium of claim 9 , wherein said at least one modified base includes a member selected from the group consisting of PPA (4-amino-1H-pyrazolo[3,4-d]pyrimidine), PPG (6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one), PPPA (4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine), PPPG (6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one), PU (5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione), PC (6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one), HOPU (5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuU (5-(4-hydroxybut-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuC (6-amino-5-(4-hydroxybut-1-ynyl)-3-hydropyrimidine-2-one), HOBuPPA (4-(4-aminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH 2 ) 2 PPPA (3-prop-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino), (NH 2 ) 2 PPPAOH (2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol), (NH 2 ) 2 BuPPAOH (4-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH 2 ) 2 PPAI (4,6-diamino-3-iodopyrazolo [3,4-d]pyrimidine), (NH 2 ) 2 PPABr (3-bromo-4,6-diaminopyrazolo[3,4-d]pyrimidine), (NH 2 ) 2 PPACl (3-chloro-4,6-diaminopyrazolo[3,4-d]pyrimidine), PPAI (4-amino-3-(iodo)pyrazolo[3,4-d]pyrimidine), PPABr (3-bromo-4-aminopyrazolo[3,4-d]pyrimidine), PPACl (3-chloro-4-aminopyrazolo[3,4-d]pyrimidine) and HOBuPPG (6-amino-3-(4-hydroxybut-1-ynyl)pyrazolo[3,4-d]pyrimidin-4-ol).
15. The computer-readable storage medium of claim 9 , wherein said identified oligonucleotide sequence has a minor groove binder (MGB) attached thereto.
16. The computer-readable storage medium of claim 15 , wherein said MGB has a formula selected from the group consisting of: wherein the subscript m is an integer of from 2 to 5; the subscript r is an integer of from 2 to 10; and each R a and R b is independently a linking group to said modified oligonucleotide, H, —OR c , —NR c R d , —COOR c and —CONR c R d wherein each R c and R d is selected from the group consisting of H, (C 1 -C 12 )heteroalkyl, (C 2 -C 12 )heteroalkenyl, (C 2 -C 12 )heteroalkynyl, (C 1 -C 12 )alkyl, (C 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl, aryl(C 1 -C 12 )alkyl and aryl.
17. A computer system for predicting the melting temperature (T m ) of an oligonucleotide sequence, comprising: a memory unit for storing nearest neighbor thermodynamic parameters; user interface means for identifying a nucleotide sequence having N bases and N-1 neighboring base pairs, wherein at least one of said N bases includes a modified base, wherein the means for identifying includes means for receiving a user entered nucleotide sequence and; processing means, coupled to the memory unit, for retrieving nearest neighbor parameters for each of the N-1 neighboring base pairs in the identified sequence from the memory unit, for calculating a melting temperature (T m ) of the identified oligonucleotide sequence using the nearest neighbor thermodynamic parameters, and for providing an output to a display or a user, wherein said output represents a T m of the identified oligonucleotide sequence, and wherein said identified sequence includes at least one modified base selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases.
18. The system of claim 17 , wherein the means for identifying includes means for receiving a GenBank GenInfo Identifier (GI) number specifying a nucleotide sequence stored at a GenBank server, the system further including means for retrieving the specified sequence from a GenBank server.
19. The system of claim 17 , wherein the means for identifying includes means for receiving a polynucleotide sequence having N or greater bases and identifying one of a portion of the received sequence and a sequence complementary to a portion of the received sequence.
20. The method of claim 1 , wherein said identified oligonucleotide sequence has attached to it one or more of a minor groove binder (MGB), a fluorophore (Fl) and a quencher (Q).
21. The method of claim 7 , where the minor groove binder is attached to the oligonucleotide via a quencher molecule.
22. The method of claim 1 , wherein identifying includes receiving a polynucleotide sequence from a sequence source.
23. The method of claim 22 , wherein the sequence source includes a GenBank server.
24. The computer readable storage medium of claim 9 , wherein the storage medium is selected from the group consisting of a floppy disk, CD and DVD.
Cooperative Patent Classification codes for this invention. Click any code to explore related patents in that topic.
June 18, 2002
May 11, 2010
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